Human Cdc25C is a protein phosphatase that dephosphorylates and activates Cdc2-cyclin B to trigger entry into mitosis. Cdc25C is itself regulated by phosphorylation. In asynchronously growing HeLa cells, we have determined that serine 216 is the major site of Cdc25C phosphorylation. We have isolated a protein kinase that binds to Cdc25C and phosphorylates serine 216. "he kinase binds within amino acids 20&256 of Cdc25C. This region is conserved in some Cdc25 homologues and contains a putative bipartite nuclear localization signal just downstream from serine 216. Finally, the Cdc25C-associating kinase was purified over 8000-fold from rat liver as a 36-38-kDa doublet of proteins. Progression through the eukaryotic cell cycle involves the sequential activation of cyclin-dependent kinases (Cdks)' (reviewed in Refs. 1 and 2). As the name implies, activation of a Cdk is dependent upon its association with a cyclin regulatory subunit. Cdk-cyclin complexes can also be regulated by reversible phosphorylation, which helps to ensure the proper timing of activation during the cell cycle (reviewed in Ref. 3). Entry of cells into mitosis is regulated in part by the activity of the Cdc2-cyclin B complex, which serves as a paradigm for the Cdk family. Cyclin B is synthesized and associates with Cdc2 in the cytoplasm beginning in S-phase and continuing throughout G,-phase (4). In higher eukaryotic cells, cyclin B association induces the rapid phosphorylation of Cdc2 on three sites. Phosphorylation on threonine 161 is required for kinase activity, whereas phosphorylation on threonine 14 and tyrosine 15 suppresses kinase activity (5-12). I t is the phosphorylation of threonine 14 and tyrosine 15 that maintains Cdc2-cyclin B in an inactive state. The kinase responsible for phosphorylating threonine 14 has not been identified, but two kinases, Weel and Mikl, have been identified that regulate the phosphorylation of tyrosine 15 (7-9, 13-15). Dephosphorylation of threonine 14 and tyrosine 15 is the final step in the activation of Cdc2-cyclin B and is required for entry of cells into mitosis (6, 314-362-7463. Dephosphorylation and activation of Cdc2-cyclin B is dependent upon the Cdc25 phosphatase. Cdc25 was first identified as a cell cycle mutant in Schizosaccharomyces pombe that acts antagonistically to Weel The Cdc25C protein is present throughout the cell cycle in higher eukaryotes, whereas its substrate (Cdc2-cyclin B) accumulates throughout the S-and G,-phases of the cell cycle Phosphorylation has been found to directly activate the enzymatic activity of Cdc25C 3046